Regulation of gene expression is fundamental to biology, and alterations in gene expression are a frequent cause of human disease. Gene regulation is typically investigated at the level of transcription, yet there is a growing recogniton of consequential post-transcriptional modulation of gene expression. In many eukaryotes, including animals, microRNAs (miRNAs) direct much of post-transcriptional regulation. MicroRNAs are short, non-coding, regulatory RNAs that post-transcriptionally repress gene expression by basepairing to target messenger RNAs (mRNAs). In humans, miRNAs contribute to a wide variety of biological pathways, moreover, mutations perturbing miRNAs, or their targeting, are implicated in a growing number of human diseases, including a variety of forms of cancer. Since their initial discovery, much has been learned about the biogenesis, regulation and mode of action of miRNAs, however, our knowledge is incomplete and the identification of novel factors involved in miRNA biology will help us better understand how these important regulatory molecules function in humans. Our first aim is to identify new protein factors involved in miRNA biology; our approach uses RNAi to inhibit each human gene and a novel cell-based screening strategy to identify genes whose inhibition alters miRNA function. In our second aim, we focus on improving our ability to identify the target mRNAs for each miRNA, this remains a fundamental question in miRNA biology, both to better understand the mechanisms of miRNAs as a class, and to understand the biological functions of individual miRNAs. Despite the increasing sophistication in computational approaches to miRNA target prediction, continued progress is limited by the availability of suitable experimental techniques and data to validate and refine models. Because state-of-the-art predictions contain many errors, yet are of great utility and widely-used, we are motivated to design an improved experimental framework for target identification; our method allows the high-throughput assessment of miRNA target sites in a minimally perturbed endogenous cellular environment. The increasingly widespread recognition of the impact of miRNAs on many fields of biology suggests that such efforts could have broad applicability.